Fig 1.
Epidermal cell size under condition of salt stress.
Two different hybrids, namely the salt-sensitive Lector (A) and the relatively NaCl-resistant SR03 (B), were raised in the absence (left, control) and presence (right, 100 mM NaCl) of an 8-day NaCl stress treatment. The size of the epidermal cells was measured in the youngest expanding leaf in a region that had emerged from the sheaths. Epidermal cell length (A), epidermal cell width (B) and epidermal cell surface area (C) are shown. The data represent the means of four biological replicates (n = 4), each run in triplicate ± SE. Sizes of 12 cells per technical replicate were quantified. Asterisks indicate significant mean differences between salt treatments and controls (*p ≤ 0.05 and **p≤ 0.01; ns = not significant; t-test). Photo in (A) amended from Geilfus [37].
Fig 2.
Capacity of the epidermal cell wall to expand.
The capacity of the epidermal cell wall to expand was measured by using frozen-thawed epidermal strips of the youngest expanding leaf in a region that had emerged from the sheath. The maximal capacity of the epidermal cell walls to expand as measured with a linear variable differential transducer was plotted in millimetres over time. Light-grey, sensitive hybrid Lector; dark-grey, resistant hybrid SR03. Extension kinetics of salt-stress treatments were normalised by the corresponding control treatments. Representative kinetics of 8 equivalent recordings.
Fig 3.
Effect of NaCl stress on the apoplastic pH in the leaf epidermal apoplast.
In planta quantification of pH in the epidermal leaf apoplast in a region that had emerged from the sheaths. Light-grey: control; dark-grey: 100 mM NaCl, duration: 8 days. Lector: sensitive hybrid; SR03: resistant maize hybrid. Measurements were conducted on 45 different plants, with 5 regions of interest. The data represent means ± SE. Asterisks indicate significant mean differences between salt treatment and control with respect to the proton concentration (*p ≤ 0.001; ns = not significant; t-test).
Fig 4.
Western blot analysis of β-expansins in the epidermal cell wall of salt-sensitive Lector.
(A) Coomassie-stained controls of the Western blot shown in (B) indicate equivalent protein levels in all lanes. c, control; salt, 8-d 100 mM NaCl treatment. Numbers 1–3 indicate biological replicates. (B) Western blot detection of β-expansins in expanding leaves of salt-treated (100 mM NaCl) and control plants. β-expansin bands appear between 40 and 25 kDa. c, control; salt, 8-d 100 mM NaCl treatment. Numbers 1–3 indicate biological replicates. (C) Densitometric analysis (TINA 2.08 software) of Western blot bands shown in (B). Bands were plotted as the average of the optical density (OD). Asterisks indicate significant difference (*p ≤ 0.01). (D) Specificity test of immunochemical β-expansin labelling. To exclude that unspecific signals were erroneously detected by the anti-peptide antibody (anti-ZmExpB), the antibody was pre-incubated with the epitope peptide before 1D Western blotting. A molar ratio of antibody to added peptide of 2:1 almost completely blocked WB labelling (signal intensity: 1513 density/mm2-background). A molar ratio of antibody to added peptide of 5:1 reduced the labelling (signal intensity: 4028 density/mm2-background). Under normal conditions without pre-incubation of the antibody with the epitope peptide (ratio of 1:0 = non-adsorbed antibody), the signal intensity was 8413 density/mm2-background.
Fig 5.
Western blot analysis of β-expansins in the epidermal cell wall of salt-resistant SR03.
(A) Coomassie-stained controls of the Western blot shown in (B) indicate equivalent protein levels in all lanes. c, control; salt, 8-d 100 mM NaCl treatment. Numbers 1–3 indicate biological replicates. (B) Western blot detection of β-expansins in expanding leaves of salt-treated (100 mM NaCl) and control plants. β-expansin bands appear between 45 and 25 kDa. c, control; salt, 8-d 100 mM NaCl treatment. Numbers 1–3 indicate biological replicates. (C) Densitometric analysis of Western blot bands shown in (B). Bands were plotted as the average of the optical density (OD). ns = not significant.
Fig 6.
Model summarising the biophysical effects of NaCl stress on the leaf epidermis and its cell wall.
Treatment of maize plants for 8 days with 100 mM NaCl stress causes a size reduction of the epidermal cells derived from growth-inhibited maize leaves. This might be attributable to the epidermal cell walls being stiffer because of the reduced abundance of cell-wall-loosening β-expansin proteins. Under stress, the more resistant maize variety acidifies its epidermal apoplast to a pH range that is more favourable for acid growth and the activation of wall-loosening expansin proteins. In good agreement with this observation, the more resistant variety has a better capacity for epidermal cell expansion. The salinity-induced cell wall rheological modifications of the epidermis emphasize a contribution of the load-bearing epidermis in restricting the expansion of the entire leaf, ultimately contributing to the salinity-induced growth reduction.